WO2016057536A1

WO2016057536A1 - Methods for preparing donor tissue for transplantation

Info

Publication number: WO2016057536A1
Application number: PCT/US2015/054274
Authority: WO
Inventors: Denise L. Faustman
Original assignee: Myostar LLC
Current assignee: Myostar LLC
Priority date: 2014-10-06
Filing date: 2015-10-06
Publication date: 2016-04-14
Anticipated expiration: 2017-04-06
Also published as: WO2016057536A8

Abstract

The present invention relates to methods, compositions, and kits for preparing donor tissue for transplantation. In particular, the present invention relates to preparing donor tissue for transplantation by contacting the donor tissue with an enzyme (e.g., papain) that cleaves MHC Class I antigens from the surface of the donor tissue. The donor tissue may be contacted with a solution (e.g., Belzer UW solution) that contains the enzyme (e.g., at a temperature in the range of about 4°C to about 40°C).

Description

METHODS FOR PREPARING DONOR TISSUE FOR TRANSPLANTATION

Field of the Invention

The field of the present invention relates to tissue transplantation.

Background

Organ transplantation is the preferred treatment for most patients with chronic organ failure. Transplantation of cells, tissues and organs has become common and is often a life-saving procedure. However, their application is limited by the medical/surgical suitability of potential recipients, an increasing shortage of donors, and premature failure of transplanted organ function. More than 40,000 kidney, heart, lung, liver and pancreas transplants are performed in the United States each year (Abbas et al., 2000). Unfortunately, there are many more transplant candidates than there are donors. To overcome this shortage, a major effort is being made to learn how to use xenografts, i.e., grafts transplanted between individuals of different species. While progress is being made in this field, most transplants are allografts, i.e., grafts transplanted between two genetically different individuals of the same species. An allogeneic transplant, while typically more successful than a xenogeneic transplant, must surmount numerous obstacles to avoid transplant failure.

Of central importance in the immunological response is the major histocompatability complex (MHC), which is a set of cell surface molecules that controls a core part of the immune system in all vertebrates and determines the compatibility of donors for organ transplant. In particular, MHC class I antigens are primarily responsible for the failure of tissues, e.g., cells, organs, or parts of organs, that are transplanted from one mammal (donor) to another (host). The MHC class I proteins are expressed in essentially all nucleated cells of the body and are a key element in the immune system's ability to distinguish between "self" molecules and "foreign" (non-self) molecules. In response to foreign molecules, such as viral proteins, peptides enfolded by the MHC class I proteins are transported to the cell surface, where the viral peptide/MHC protein complex is displayed as a surface antigen. Circulating cytotoxic T lymphocytes (CTLs) having the appropriate specificity recognize the displayed MHC class I antigen as foreign and proceed, through activation and a complex lytic cascade, to kill the infected cell.

Recognition of donor MHC class I antigens as foreign (non-self) by host CTLs occurs not only in xenogeneic transplants, but also in allogeneic transplants. The specificity of the T cell receptors on CTLs and other T cells that bind to MHC class I antigens is such that a single amino acid difference in the structure of a MHC antigen can be detected as foreign, leading to an immune response. The MHC class I proteins are expressed from highly polymorphic gene segments with great diversity in the intrinsic coding sequences. Thus, between genetically unrelated individuals the incidence of MHC class I protein matching is only about 1 in 40,000, and the transplantation reaction is only avoided in the case of isogeneic grafts, i.e., the transplantation of tissues between individuals having a high degree of genetic identity, such as between identical twins or from a parent to first generation offspring. See, Roitt et al., Immunology (2nd ed. 1 989), Chapt. 24, pp. 24.1 -24.1 0.

Several methods have been devised to overcome MHC class I antigen recognition and its consequences for transplanted donor tissue, including the use of immunosuppressive drugs, such as cyclosporin A, the masking or blocking of MHC class I antigens on donor tissue, for example with monoclonal antibodies against the MHC class I antigens (see, e.g., U.S. Pat. No. 5,283,058), and the preparation of donor tissues in transgenic animals that have been genetically altered to decrease or eliminate MHC class I expression (see, e.g., Li et al., Transplantation, 55:940-6 (1993) ; and Coffman et al., J. Immunol., 151 :425-35 (1993)). Each of these methods can be effective in overcoming rejection or prolonging the survival of donor tissues, but they also have potential drawbacks. For example, they may cause serious side effects, such as renal failure and hypertension, they may render the host susceptible to infection and tumor growth, or they may be generally labor intensive.

In light of these factors, there remains an unmet need in the field of organ transplantation for methods for preparing cells, tissues, and organs for transplantation that achieve greater functionality and widespread application to a diverse array of donor tissues.

Summary of the Invention

The invention features methods, compositions, and kits for preparing donor tissue for transplantation by contacting the tissue with an enzyme that cleaves MHC class I antigens from the surface of the tissue.

In a first aspect, the invention features a method for preparing donor tissue for transplantation including contacting the donor tissue at a temperature of about 22^SC or less with a composition including an enzyme that cleaves MHC class I antigens from the surface of the donor tissue (e.g., a cysteine protease, such as papain). In some embodiments of the first aspect, the contacting reduces or inhibits activation or stimulation of immune cells (e.g., T cells). For example, the contacting can occurs at a temperature in the range of about 0^SC to about 15^SC, such as about 4^SC.

In a second aspect, the invention features a method for preparing donor tissue for transplantation including contacting the donor tissue with a composition containing an organ storage solution and an enzyme that cleaves MHC class I antigens from the surface of the donor tissue (e.g., a cysteine protease, such as papain). In some embodiments of the second aspect, the donor tissue is contacted at a temperature of about 40^SC or less (e.g., a temperature in the range of about 22^SC to about 40^SC, such as about 37^SC).

In a third aspect, the invention features a method for reducing or inhibiting a immune rejection (e.g., hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, or graft versus host disease (GVHD)) by contacting donor tissue at a temperature of about 22^SC or less (e.g., a temperature of about 0°C to about 15°C) with an enzyme (e.g., a cysteine protease, such as papain) that cleaves MHC class I antigens from the surface of the donor tissue (e.g., the method involves treating donor tissue in situ in a host recipient after transplantation of the donor tissue).

In a fourth aspect, the invention features a method for treating GVHD in a host recipient in need thereof by contacting transplanted donor tissue in situ in the host recipient with an enzyme (e.g., a cysteine protease, such as papain) that cleaves MHC class I antigens from the surface of the donor tissue. In some embodiments of the fourth aspect, the host recipient exhibits one or more symptoms of GVHD selected from the group consisting of abdominal pain, cramping, diarrhea, fever, jaundice, skin rash, vomiting, weight loss, dry eyes, dry mouth, hair loss, hepatitis, lung disorders, and digestive tract disorders. In embodiments of the first, second, third, and fourth aspects of the invention, the contacting reduces or inhibits activation or stimulation of immune cells (e.g., T cells). For example, the contacting reduces or inhibits transplant rejection (e.g., hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, and GVH D) of the donor tissue by a host recipient, e.g., when the host recipient has a human leukocyte antigen (HLA) type that does not match the H LA type of the donor tissue (e.g., a host recipient that shares at least 4/6 HLA alleles (or 5/6 HLA alleles) with the donor tissue.

In several embodiments of the first, second, third, and fourth aspects of the invention, the enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof (e.g., the enzyme is selected from papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, and combinations thereof). In preferred embodiments, the enzyme is papain.

In other embodiments of the first, second, third, and fourth aspects of the invention, the donor tissue is an autograft, a syngeneic graft, an allograft, or a xenograft. In still other embodiments of the first and second aspects, the donor tissue is from a mammal that is the same species as the host recipient or from a mammal that is a different species from that of the host recipient (e.g., the donor tissue can be from , and/or the host receipt can be, a human). In several embodiments, the donor tissue is genetically modified prior to transplantation.

In some embodiments of the first, second, third, and fourth aspects of the invention, the donor tissue is or includes a cell, a tissue, or an organ. In particular embodiments of the first and second aspects, the donor tissue is a whole or a partial organ. In some embodiments of the first and second aspects, the cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet), cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, hepatocyte, hematopoietic cell, bone cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof. In other embodiments, the tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof (e.g., the tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof). In various embodiments, the organ is selected from the group consisting of nervous system , brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood (e.g., red blood cells (e.g., blood cells of the A, B, AB, and 0 subgroups), white blood cells), prostate, cornea, fetal organs, and combinations thereof. In an embodiment, the cell, tissue, or organ is viable tissue (e.g., non-fixed tissue). In other embodiments, the cell, tissue, or organ is non-viable tissue (e.g., fixed tissue). The donor tissue can be made non-viable before or after contacting with the enzyme that cleaves MHC class I antigens from the surface of the donor tissue (e.g., a cysteine protease, such as papain). For example, the non-viable tissue is tissue that has been treated to remove all or a portion of the living cells but to leave the underlying structure (e.g., extracellular matrix or structural material (e.g., collagen, cartilage, and bone) intact). Examples include corneas, cartilage, heart values, skin, bone, and organ structures that have been treated to remove all or a portion of the living cells.

In an embodiment of the first, second, third, and fourth aspects of the invention, prior to contacting the donor tissue, the enzyme (e.g., a cysteine protease, such as papain) can be activated by incubating the enzyme at a temperature in the range of about 22^SC to about 37^SC. For example, the enzyme can be activated in the presence of a reducing agent (e.g., cysteine) and/or in the presence of EDTA.

In other embodiments of the first, second, third, and fourth aspects of the invention, the donor tissue is contacted with the enzyme (e.g., a cysteine protease, such as papain) for a period of about 5 minutes to about 24 hours (e.g., about 1 hour to about 5 hours, such as about 3 hours).

In an embodiment of the first, second, third, and fourth aspects of the invention, the composition includes, in addition to the enzyme (e.g., a cysteine protease, such as papain), an organ storage solution (e.g., Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof). In other embodiments, the organ storage solution includes one or more (e.g., two, three, four, five, or more (e.g., at least two)) components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG). In particular embodiments, the organ storage solution includes potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and HES (e.g., about 10 mM to about 200 mM (e.g., about 100 mM) potassium lactobionate, about .25 mM to about 100 mM (e.g., about 25 mM) potassium phosphate, about .5 mM to about 50 mM (e.g., about 5 mM) magnesium phosphate, about 3 mM to about 100 mM (e.g., about 30 mM) raffinose, about 1 mM to about 50 mM (5 mM) adenosine, about 0.3 mM to 30 mM (e.g., about 3 mM) glutathione, about 0.1 mM to about 10 mM (e.g., about 5 mM) allopurine, and about 1 g/L to about 100 g/L (e.g., about 50 g/L) hydroxyethyl starch). In particular embodiments, the composition includes Belzer-UW solution and papain.

In an embodiment of the first, second, third, and fourth aspects of the invention, the method further includes the step of transplanting the donor tissue into the host recipient (e.g., before MHC class I antigens are re-expressed on the surface of the donor tissue). For example, the transplanting can occur within a period of about 1 minute to about 48 hours after the contacting (e.g., about 1 hour to about 5 hours after the contacting, such as about 3 hours). In various embodiments, the method further includes bathing or perfusing the donor tissue (e.g., prior to transplantation) with a perfusing liquid (e.g., the perfusing liquid is or includes saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, therapeutic agents, or combinations thereof). The method may further include the step of washing the donor tissue prior to transplanting the donor tissue into the host recipient and/or the step of transplanting a second donor tissue into the host recipient (e.g., the second donor tissue can be prepared for transplantation by contacting the second donor tissue with an enzyme that cleaves MHC class I antigens from the surface of the second donor tissue (e.g., a cysteine protease, such as papain). In particular embodiments, the contacting is at a temperature of about 22^SC or less.

In another embodiment of the first, second, third, and fourth aspects of the invention, the method further includes administering an immunosuppressive drug or an immunomodulatory cell (e.g., a Treg) to the host recipient. Examples of immunosuppressive drugs include cyclosporin A, tacrolimus, sirolimus, OKT3, a corticosteroid, daclizumab, basiliximab, azathioprene, mycophenolate mofetil, methotrexate, 6- mercaptopurine, anti-T cell antibodies, cyclophosphamide, leflunamide, brequinar, ATG, ALG, 1 5- deoxyspergualin, LF15-0195, bredinin, and combinations thereof. The immunosuppressive drug and/or the immunomodulatory cell can be administered before, simultaneously with, or after transplantation of the donor tissue to the host recipient.

In yet another embodiment of the first, second, third, and fourth aspects of the invention, the contacting is performed in situ in the host recipient (e.g., before or after (preferably after) the donor tissue has been transplanted). For example, the method inhibits or reduces immune rejection of a previously transplanted donor tissue. The in situ method can be performed, e.g., by bathing cells of the donor tissue in the host recipient with a composition that includes the enzyme or by perfusing the donor tissue (e.g., the entire organ), the transplant donor (e.g., prior to harvest of the donor tissue), or the host recipient, with a composition that includes the enzyme. For example, a composition that includes an enzyme (e.g., a cysteine protease, such as papain) could be administered to the transplant donor and/or the host recipient, e.g., by parenteral administration, oral administration, subcutaneous administration, intravenous administration, intraarterial administration, or topical administration (e.g., the composition could be administered using a perfusion machine, such as a dialysis machine). Alternatively, or in combination, the enzyme (e.g., a cysteine protease, such as papain) can be contacted to the donor tissue at the time of harvest from a transplant donor.

In a fifth aspect, the invention features a method for reducing or inhibiting immune rejection (e.g., hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, and GVHD) in a host recipient by transplanting a donor tissue treated according to the method of any one of the first, second, third, and fourth aspects of the invention and embodiments thereof into the host recipient. In some embodiments of the fifth aspect, the donor tissue is or includes a cell that is administered by parenteral administration, oral administration, subcutaneous administration, intravenous administration, intraarterial administration, or topical administration.

In a sixth aspect, the invention features a composition for preparing donor tissue for

transplantation. The composition includes an enzyme (e.g., a cysteine protease, such as papain) that cleaves MHC class I antigens and an organ storage solution (e.g., Belzer UW solution).

In a seventh aspect, the invention features a composition for transplantation that includes donor tissue, an enzyme (e.g., a cysteine protease, such as papain) that cleaves MHC class I antigens, and an organ storage solution (e.g., Belzer UW solution). In embodiments of the sixth and seventh aspects of the invention, the enzyme is a proteolytic enzyme, glycosidase, proteinase, or a combination thereof. In particular embodiments of the sixth and seventh aspects, the enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5- thiocyanobenzoate, or endopeptidase, or a combination thereof. In preferred embodiments, the enzyme is papain.

In some embodiments of the sixth and seventh aspects of the invention, the donor tissue is from a mammal (e.g., a human). In some embodiments, the donor tissue is genetically modified. In various embodiments, the donor tissue includes a cell, a tissue, or an organ (e.g., a whole or a partial organ). In some embodiments, the cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet), hepatocyte, cardiac cell, lung cell (e.g., alveolar cell) , genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, bone cell, hematopoietic cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), a brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof. In other embodiments, the tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof (e.g., the tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof). In still other embodiments, the organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood (e.g., red blood cells (e.g., blood cells of the A, B, AB, and 0 subgroups), white blood cells), prostate, cornea, fetal organs, and combinations thereof.

In embodiments of the sixth and seventh aspects of the invention, the enzyme is an activated enzyme (e.g., the enzyme is activated by incubating the enzyme at a temperature in the range of about 22^SC to about 37^SC and/or in the presence of a reducing agent (e.g., cysteine) and/or in the presence of EDTA).

In some embodiments of the sixth and seventh aspects of the invention, the organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof. In other embodiments, the organ storage solution includes one or more (e.g., two, three, four, five, or more (e.g., at least two)) components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG). In particular embodiments, the organ storage solution includes potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and HES (e.g., about 10 mM to about 200 mM (e.g., about 100 mM) potassium lactobionate, about .25 mM to about 100 mM (e.g., about 25 mM) potassium phosphate, about .5 mM to about 50 mM (e.g., about 5 mM) magnesium phosphate, about 3 mM to about 100 mM (e.g., about 30 mM) raffinose, about 1 mM to about 50 mM (5 mM) adenosine, about 0.3 mM to 30 mM (e.g., about 3 mM) glutathione, about 0.1 mM to about 10 mM (e.g., about 5 mM) allopurine, and about 1 g/L to about 100 g/L (e.g., about 50 g/L) hydroxyethyl starch). In particular embodiments, the composition includes Belzer-UW solution and papain. In another embodiment, the enzyme retains activity in the organ storage solution.

An eight aspect of the invention features a kit for preparing donor tissue for transplantation. The kit includes an enzyme (e.g., a cysteine protease, such as papain) that cleaves MHC class I antigens from the surface of the donor tissue and an organ storage solution (e.g., Belzer UW solution). In an embodiment of the eighth aspect, the enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof. In some embodiments, the enzyme is papain or a papain-like enzyme,

endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu- specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N- chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, or endopeptidase, or a combination of such enzymes. In preferred embodiments, the enzyme is papain. In various embodiments, the organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof. In other embodiments, the organ storage solution includes one or more (e.g., two, three, four, five, or more (e.g., at least two)) components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha- ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG). In particular embodiments, the organ storage solution includes potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES (e.g., about 10 mM to about 200 mM (e.g., about 100 mM) potassium lactobionate, about .25 mM to about 100 mM (e.g., about 25 mM) potassium phosphate, about .5 mM to about 50 mM (e.g., about 5 mM) magnesium phosphate, about 3 mM to about 100 mM (e.g., about 30 mM) raffinose, about 1 mM to about 50 mM (5 mM) adenosine, about 0.3 mM to 30 mM (e.g., about 3 mM) glutathione, about 0.1 mM to about 10 mM (e.g., about 5 mM) allopurine, and about 1 g/L to about 100 g/L (e.g., about 50 g/L) hydroxyethyl starch). In particular embodiments, the composition includes Belzer-UW solution and papain. In another embodiment, the enzyme retains activity in the organ storage solution.

In an embodiment of the eighth aspect, the enzyme and the organ storage solution are in one or more containers in the kit. In some embodiments, the enzyme and the organ storage solution are in the same container. In an alternative embodiment, the enzyme and the organ storage solution are in different containers. Brief Description of the Drawings

Figure 1 A is a schematic diagram of the two polypeptide chains of HLA class I. Figure 1 B is a table summarizing papain adaptation to organ transplant conditions.

Figure 2 is a graph showing that papain can have proteolytic cutting activity in Belzer-UW solution compared to distilled water (DW). Casein cleavage by papain was quantified either dissolved in distilled water (solid squares) or dissolved in Belzer-UW solution (solid triangles) with a dose response curve of papain at a stock concentration of 0.05mg/ml.

Figures 3A and 3B are graphs showing the impact of papain usage at 4^SC versus 37^SC in distilled water (Figure 3A) and Belzer-UW Solution (Figure 3B). As shown in Figure 3A, in distilled water papain activation at 37^SC followed by cooling to 4^SC for casein cleavage can be made equivalent to papain activation under the Regular Protocol (solid squares) if exposed to casein for 1 hour (solid diamond) or for 3 hours (solid circle). As shown in Figure 3B, in Belzer-UW solution, papain can be shown to have proteolytic cleavage activity even when used at 4^SC if co-incubated with casein for 3 hours (solid dots), but not after 1 hour (solid triangle). The represented p values show no difference between the ability of papain to cleave the casein test substrate when dissolved in distilled water or Belzer-UW solution.

Figure 4A is a graph showing the impact of papain usage at 4^SC versus 37^SC in Belzer-UW solution. Figure 4B is a graph showing a characteristic set of flow cytometric diagrams showing the gradual and complete removal of highly expressed HLA classes I from the surface of PBLs when exposed to graded papain concentrations. The percentages of PBLs still expressing H LA class I for each papain concentration are depicted in the histograms.

Definitions

The term "proteolytic activity," as used herein, refers to the cleavage activity of a substrate by an enzyme. In particular embodiments, the term refers to the enzymatic cleavage by endopeptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endo-peptidase activity. In exemplary embodiments, the term is meant to refer to the specific activity of cysteine proteases (e.g., papain (e.g., EC 3.4.22.2)) and papain-like cysteine proteases, having, e.g., the ability to cleave at recognition sites corresponding to Xaa - HYD - Arg/Lys - Xaa (not Val) - Xaa, in which the "Xaa" is any amino acid and "HYD" is a hydrophobic residue, such as Ala, Val, Leu, lie, Phe, Trp, or Tyr. Other cysteine proteases include cathepsin B, cathepsin C, cathepsin F, cathepsin H, cathepsin L1 , cathepsin L2, cathepsin L3, cathepsin 0, cathepsin S, cathepsin Z, and cathepsin X. "Non-specific proteolytic activity" is meant to refer to cleavage activity that is not directed to a specific cleavage site. "Specific proteolytic activity" is meant to refer to cleavage activity that is directed to a specific cleavage site.

The term "about," as used herein, means ±10% of the recited value.

The term "administering" as used herein, refers to a method of delivering donor cells in solution to a recipient host by any method of administration known in the art. Examples of administration methods include, but are not limited to, parenteral administration, oral administration, subcutaneous administration, intravenous administration, intraarterial administration, and topical administration.

The term "donor tissue" as used herein, refers to any cell, tissue, or organ (e.g., a whole or partial organ) that is transplanted from one subject (i.e., a donor) into a transplant patient. Non-limiting examples of donor tissue include all or a portion of nervous system , brain (e.g., neuronal tissue), spinal cord, heart, heart valves, lung, kidney, liver, pancreas (e.g., islet cells and islets of Langerhans), gall bladder, urinary tract, adrenal gland, thymus, spleen (e.g., Hox1 1 + cells), lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood (e.g., red blood cells (e.g., blood cells of the A, B, AB, and 0 subgroups), white blood cells, serum, and plasma), blood vessels (e.g., veins and arteries), prostate, cornea, skin, bone marrow, stem cells, mesenchym cells, endothelium , epithelium , a fetal organ, cartilage, and combinations thereof.

The term donor tissue includes allogeneic and xenogenic tissue.

The term "enzyme that cleaves MHC class I antigens," as used herein, refers to any

macromolecular biological catalyst capable of chemically removing MHC class I antigens from the surface of donor tissue (e.g., by proteolytic activity) in preparation for transplantation. The enzyme is used in an amount and for a sufficient time period to significantly attenuate the host's immune response to the donor tissue, in comparison to the host's immune response to untreated donor tissue. Preferably the mean cell density of MHC class I antigens treated with such enzymes will be reduced by at least 10%, preferably by at least 50%, preferably by at least 75%, and most preferably by at least 95% or more compared to untreated tissue. Examples of such enzymes include papain, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5- thiocyanobenzoate, endopeptidase, or a combination thereof. In particular, the enzymatic reagent for use in the method of this invention is a cysteine protease, such as papain.

The term "graft versus host disease" or "GVHD" as used herein, refers to a condition that occurs following the transplantation of a donor tissue into a transplant patient. Generally, functional immune cells in the transplant tissue recognize the recipient as "foreign" and stimulate or activate immune cells (e.g., T-cells) that originate from the donor tissue, which then target the recipient's tissue. GVHD can be acute or chronic depending on the timing of the onset of, and the type of, symptoms of the disease.

The term "host recipient" or "transplant patient," as used interchangeably herein, refers to any animal (e.g., a mammal, such as a human) that receives via administration or transplant cells, tissues, or organs from a donor animal (e.g., a mammal, such as a human).

The term "immunosuppressive drug," as used herein, refers to an agent that suppresses or inhibits the immune system of a patient to which the agent is administered (e.g., the humoral and/or cell- mediated immune responses in a patient). Non-limiting examples of immunosuppressive agents include steroids, glucocorticoids, corticosteroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisone, prednisone, prednisolone, azathioprine, FK-506, cyclosporin G, leflunomide, mizoribine, berquinar sodium , azaspirane, rapamycin, 15-deoxyspergualin, methotrexate,

mercaptopurine, mycophenolic motefil, tacrolimus, sirolimus, thalidomide, and methoxsalen.

The term "mammal," as used herein, refers to a human, non-human primate, or other mammal, such as but not limited to dog, cat, mouse, rat, horse, rabbit, cow, pig, goat, monkey, and sheep.

The term "organ storage solution," as used herein, refers to a sterile preservation medium designed for flushing and storage of donor tissue (e.g., cells, tissues, and organs) at the time of, or subsequent to, their removal from the organ donor in preparation for storage, transportation and eventual transplantation into a host recipient. Such compositions aim to preserve the cellular function of the desired donor tissue (e.g., cells, tissue, and organs) ; seek to mimic the intracellular electrolyte balance of mammalian cells; and reduce the redox changes associated with organ hypoxia during preservation.

The term "reduce or inhibit activation or stimulation of immune cells", as used herein, refers to a decrease of at least 10% or 15%, more preferably by at least 20% or 25%, and most preferably by at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more, in at least one measured activity of a stimulated immune cell (e.g., an activated T cell). Preferably at least 2, 3, 4, 5, or 6 different measured stimulated immune cell activities are decreased. Examples of stimulated T cell activities include the production of specific cytokines (non-limiting examples include: IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, IFN-γ, GM-CSF, TGF-β, and TNF-β), the production and release of toxic proteins (e.g., perforin and granzymes), the expression of cell surface receptors (non-limiting examples include insulin receptor, IL-2 receptor, HLA-DR, VLA-1 , VLA-2, VLA-3, VLA-4, and VLA-5), and cell division (i.e., clonal expansion). Several assays to measure immune cell activity include, but are not limited to:

immunoassays for the measurement of the levels of cytokines produced by activated immune cells (e.g., IL-2 and IFN-γ) ; mixed lymphocyte reaction (MLR) assays (Ginsburg and Sachs, J. Cell Physiol. 66:199- 219, 1965) ; and [H³]-thymidine incorporation assays for the measurement of clonal expansion.

The term "symptoms of graft versus host disease" or "symptoms of GVHD", as used herein, refers to one or more clinical symptoms present in a patient having GVH D. Examples of symptoms of GVHD include, but are not limited to: abdominal pain, cramping, diarrhea, fever, jaundice, skin rash, vomiting, weight loss, dry eyes, dry mouth, hair loss, hepatitis, lung disorders, and digestive tract disorders.

The term "transplantation," as used herein, refers to the grafting of cells, tissues, and/or organs

(e.g., a whole or partial organ) from the body of an individual (e.g., a donor) to a different place with the same or a different individual (e.g., a host recipient). Transplantation can be grafting of cells, tissues, and/or organs from one area of the body to another. Transplantation of cells, tissues, and/or organs between genetically dissimilar animals of the same species is termed allogeneic transplantation.

Transplantation of animal organs into humans is termed xenotransplantation.

The term "transplant rejection," as used herein, refers to the functional and structural deterioration of a donor cell, tissue, or organ that has been transplanted into a host recipient due to an active immune response mediated by the host recipient. Rejections include the partial or complete destruction of cell(s), tissue(s), or organ(s) of the transplanted donor tissue in the host recipient.

Detailed Description of the Invention

The present invention features methods, compositions, and kits for preparing donor tissue for transplantation. Expression of MHC class I antigens is a well-known barrier to organ transplantation. Due to the high degree of polymorphism, meticulous matching of donor MHC class I antigens to the recipient to thwart organ rejection is often necessary even in combination with the administration of immunosuppressive drugs to the recipient. For instance, human leukocyte antigen (HLA, the human version of the MHC) matching of unrelated donors significantly improves the success rate of allogeneic transplants when donor and recipient cells share at least 4/6 HLA markers in common, more preferably 5/6 markers, and most preferably 6/6 markers. Methods of the present invention may be particularly useful when HLA matching is not possible (e.g., in transplant patients in which 4/6 or 5/6 HLA markers can be matched). Another approach to tackle transplant rejection is not only to target host T cells through immunosuppressive drugs, but to also remove donor M HC class I structures from the donor organ. Enzymes selected for this method are those capable of cleaving MHC class I antigens. To date, donor MHC class I removal strategies have been applied successfully to cellular transplants, such as insulin secreting islets of Langerhans or fetal pig neuron transplants in murine models. However, application of this technology in the whole organ transplant setting has not been previously tackled. We have discovered that papain, a selective MHC class I removing enzyme, and other enzymes having similar proteolytic activity in cleaving MHC class I from the cell surface (e.g., other cysteine proteases), can be used to modify donor cells or whole organ tissue under desirable transplant conditions. Described herein are methods, compositions, and kits using this invention to prepare donor tissue for transplantation.

The methods, compositions, and kits described herein can be used to reduce or inhibit immunological rejection of donor tissue by a recipient that can lead to the failure of the donor tissue to engraft. These types of immunological rejection that can be addressed by the methods, compositions, and kits described herein include hyperacute rejection, acute vascular rejection (including accelerated humoral rejection and de novo acute humoral rejection), chronic rejection, rejections due to non- adherence, and graft versus host disease (GVHD). In particular, GVHD is the most common cause of post-transplant morbidity and mortality following donor tissue transplantation. GVHD appears in both chronic forms, which occur within 100 days of transplant, and acute or fulminant forms, which occur after 100 days of transplant. The methods of the present invention may be used to reduce or inhibit either form of GVHD following receipt by the transplant patient of a donor tissue. Another example of a T-cell mediated immunological response in the recipient host that may be minimized by the methods described herein includes transplant rejections in the context of skin grafts. For example, the treatment methods can be used to reduce or inhibit a first-set rejection, which occurs when a skin allograft is initially accepted by the recipient host and is then rejected about 1 0 days after transplantation, as well as a second-set rejection, which occurs when a second skin graft from the same donor is rejected more rapidly (e.g., within about 6-8 days).

The present invention may be used to treat donor tissues prior to storage or prior to

transplantation of the donor tissue into a host recipient. The tissues that are advantageously treated in accordance with the teachings herein can be any tissues, e.g., cells, tissues, or organs, of benefit to a host recipient. Cells that can be treated according to the methods of the invention include, e.g., a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet), hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, skin cell, precursor cell, fibroblast, myoblast, bone marrow cell, hematopoietic cell, bone cell, islet cell (e.g, islets of Langerhans), spleen cell (e.g., Hox1 1 + cell), a brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell, spleen cell (e.g., Hox1 1 + cell), fetal cells, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, and combinations thereof.

Tissues that can be treated according to the methods of the invention include, e.g., bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof. In some embodiments, the tissue is epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof. The methods of the present invention may also be used on tissues treated or not treated for preservation (e.g., fixation). Examples of chemical fixatives that may be used include aldehydes (e.g., formaldehyde, gluteraldehyde) , alcohols (e.g., ethanol, methanol), oxidizing agents (e.g., osmium tetroxide, potassium diochromate), mercurials (B-5, Zenker's fixative), picrates, HOPE fixative, and any combination thereof.

Organs that can be treated according to the methods of the invention include, e.g., nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood (e.g., red blood cells (e.g., blood cells of the A, B, AB, and 0 subgroups), white blood cells), prostate, cornea, fetal organs, and combinations thereof.

The donor tissue can be xenogeneic, allogeneic, or syngeneic with respect to the host recipient.

The present invention also includes methods for genetically modifying or engineering donor tissue prior to transplantation into a recipient to ensure that transplant rejection does not occur. The gene therapy methods include modifying or engineering donor tissue to reduce the expression of immune-stimulating proteins or to increase the expression of proteins that modulate (.e.g, downregulate) an immune response. For example, donor tissue can be genetically modified or engineered to reduce or eliminate the expression MHC class I antigens (e.g., HLA-A, H LA-B, H LA-C, HLA-E, HLAF, and HLA-G), cytokines (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, IFN-γ, GM-CSF, TGF-β, and TNF-β), class II transactivators (CIITA), PDL1 , PDL2, toxic proteins (e.g., perforin and granzymes), cell surface receptors (e.g., insulin receptor, IL-2 receptor, HLA-DR, VLA-1 , VLA-2, VLA-3, VLA-4, and VLA-5), and

combinations thereof. Gene therapy methods for mammalian cells that may be used in the present invention to genetically modify (e.g., to ablate) genes encoding immune-stimulating proteins in the donor tissue or to modify the donor tissue to express immune-modulating proteins include, but are not limited to, transfection, transformation, virus-mediated transduction, infection with a viral vector (e.g., adenoviral vectors, retroviruses, lentiviruses, or other viral vectors), or combinations thereof. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art utilizing a retroviral particle containing RNA encoding genes that reduce expression of the MHC class I antigens on the surface of the donor tissue.

Methods of preparing donor tissue for transplantation

Enzymes

Enzymes that can be selected for use in the methods of the invention are those that are capable of cleaving MHC class I antigens (e.g., removing an MHC class I protein/peptide complex) from the surface of a cell on which it is displayed. A preferred enzyme for use in the methods of the invention is papain. Any enzyme capable of altering the display of MHC class I antigen on the surface of donor tissue sufficiently to avoid interaction with the immune system cells of the host is suitable. Preferably, the majority of the extracellular portion of the MHC class I antigen is removed from the cell after treatment with the enzyme. Any amount of MHC class I antigen that can be removed from the donor tissue will be helpful in avoiding rejection of the transplant. Treatment of the donor tissue with the enzyme (e.g., papain) substantially reduces the amount of MHC class I antigen, e.g., to a point where the immune response of the host following transplant of the donor tissue is measurably altered in comparison to the response mounted against untreated tissue. Removal of as much of the MHC class I antigens as possible without killing the tissue is desired. In particular, the methods reduce the amount of MHC class I antigen on the surface of the donor tissue cells by 10% or more, more preferably by 50% or more, preferably by 75% or more, and most preferably by 95% or more, e.g., as measured by flow cytometry using a fluorescently labeled anti-MHC class I antibody. For example, after treating donor tissue according to the methods of the invention less than 10% of the MHC class I antigen will remain on the cell surface (e.g., less than 5% of the MHC class I antigen will remain on the cell surface). Thus, about 10% to about 100% of the MHC class I antigens can be removed from the surface of the donor tissue cells by the treatment methods.

The donor tissue intended for transplant is contacted with the enzyme at a sufficient

concentration, e.g., greater than about 1 μg/ml papain, or a concentration of enzyme in the range of about 0.1 μg/ml to about 500 mg/ml (e.g., about 1 μg/ml to about 100 mg/ml, such as about 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml, 1 mg/ml, 2, mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, or 180 mg/ml). The contacting step may be performed on donor cells (e.g., cells, tissues, or organs) at the time of harvest or at the site of transplant. Furthermore, the contacting step occurs for a period of time during which cleavage of the class I antigens occurs. This result can be accomplished by bathing or perfusing the donor tissue with a solution containing the enzyme and for a period of time to allow the enzyme to react with the MHC proteins. For example, the contacting step may be performed for a period of time from , e.g., about 5 minutes to about 24 hours or more. At high enzyme concentrations, e.g., about 30-50 μg/ml papain, incubation of the donor tissue may be for even shorter periods (e.g., for a time of less than about 5 hours, such as about 3 hours), as long as the cells of the tissues are not damaged. The tissues should remain viable for transplantation. Therefore, the treatment can be adjusted, if necessary, so as to avoid killing a high percentage of donor cells during the treatment process or otherwise rendering the donor cells unsuitable for transplantation. Generally, the aim is for at least 75% of the tissue cells to remain viable after treatment with the enzyme. Preferably more than 90% of the tissue cells will remain viable after enzymatic treatment. The enzymatic treatment may be performed at a range of temperatures (e.g., a temperature in the range of about 2 °C to about 40°C). For example, the enzymatic treatment may be performed at room temperature, e.g., at about 20 Ό to 26Ό, such as about 22 °C, or at the temperature of the host recipient, e.g., a temperature in the range of about 30 °C to about 40 °C, such as about 37°C. Preferably, the temperature will be at or near conditions conducive to organ preservation and perfusion, e.g., in the range of about 2°C to about 8 Ό, such as about 4Ό. As is discussed below, the enzymatic treatment may also be performed in an organ storage solution (for example, University of Wisconsin (UW) solution or other solution, such as those described below; e.g., at a temperature in the range of about 2°C to about 40°C, such as about 4°C, about 22°C, or about 37Ό). The enzymatic treatment may also be performed for a range of incubation periods (e.g., an incubation period of about 5 minutes to about 24 hours). For example, the enzymatic treatment may be performed for about 5 minutes to 10 minutes or for longer incubation periods of about 5 hours to 24 hours (e.g., about 3 hours). Preferably, the incubation period (e.g., from about 1 hour to about 24 hours, such as about 1 hour to about 5 hours, such as 3 hours) will correspond to proteolytic activity that results in a substantial decrease in MHC class I antigens on the surface of the donor tissue (e.g., a decrease in MHC class I antigens on the surface of the donor tissue of greater than about 10%, such as a decrease in MHC class I antigens on the surface of the donor tissue of about 10% to about 90% or more, such as a decrease in MHC class I antigens on the surface of the donor tissue of greater than about 50%; all relative to untreated donor tissue). As is discussed below, organ storage solution (e.g., UW solution) may also be used over this range of enzyme incubation periods and can achieve a decrease in MHC class I antigens on the surface of the donor tissue corresponding to the recited amounts (e.g., when a cysteine protease (e.g., papain) is the enzyme).

There are several advantages to the use of enzymes, such as those described below (e.g., a cysteine protease (e.g., papain)), for treating donor tissue prior to transplantation in order to reduce or inhibit transplant rejection: (a) enzymes are comparatively inexpensive and many are commercially available in high purity with well-characterized activity and specificity; (b) enzyme reagents are in most cases very stable and can be stored for months without special handling or refrigeration; (c) enzymes can be used locally or in vitro to avoid systemic treatments; (d) the effectiveness of the enzyme treatment does not depend on permanent or constant attachment or binding to donor cell surface structures; (e) enzymatic cleavage of the transplant tissue can be used in combination with other complementary treatments or therapies (e.g. administration of immunosuppressive drugs) ; and (f) the use of enzymes is not restricted to species or alleles. Thus, the methods of the invention are adaptable to veterinary, human, and xenogeneic tissue treatment without radical modification of the procedures or reagents. Since the tissues remain viable after treatment according to this invention, expression of MHC molecules will continue, and eventually reappearance of MHC antigens on the donor tissue will occur, e.g., after transplantation. The present methods that combine an enzyme (e.g., a cysteine protease (e.g., papain)) with an organ storage solution (e.g., Belzer UW solution) are also advantageous since they achieve two simultaneous goals: the organ storage solution stabilizes and preserves the donor tissue during the time after harvesting from the donor and until transplantation into the recipient, while the enzyme reduces or inhibits graft rejection upon transplantation by cleaving MHC class I antigens from the surface of cells of the donor tissue.

Consequently, the present method may be used as part of an overall therapy including additional measures to avoid latent rejection, such as, e.g., the administration of immunosuppressive drugs, plasmaphoresis, the use of antigen blocking methods or reagents, transfection of genes that modulate the immune response (e.g., genes encoding transforming growth factor beta or interleukin 10), tolerance induction in the host by the early masking of the donor graft, and any combination thereof. In some embodiments, an immunosuppressive drug may be administered. Examples of immunosuppressive drugs include, but are not limited to, steroids, glucocorticoids, corticosteroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisone, prednisone, prednisolone, azathioprine, FK-506, cyclosporin G, leflunomide, mizoribine, berquinar sodium, azaspirane, rapamycin, 15-deoxyspergualin, methotrexate, mercaptopurine, mycophenolic motefil, tacrolimus, sirolimus, thalidomide, methoxsalen, or a combination thereof.

The present treatment methods may also include the administration of immune-modulating cells, such as Tregs (see, e.g., PCT/US2014/015101 , incorporated herein by reference).

Furthermore, donor cells treated according to the methods of the present invention may be formulated for several different routes of administration. Methods of administering isolated donor cells in solution to a recipient host may include, but are not limited to, parenteral, oral, subcutaneous, intravenous, intraarterial and topical administration. When combined with immunosuppressive drugs, the treated donor cells and immunosuppressive drug(s) may be formulated for the same route of

administration (e.g., both formulated for intravenous administration) or formulated for different routes of administration (e.g., the donor cells formulated for intravenous administration and the immunosuppressive agent(s) formulated for oral administration). In addition, the donor cells of the present invention and/or immunosuppressive agent(s) may be administered to target a specific affected tissue or region of the body (e.g., nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood (e.g., red blood cells (e.g., blood cells of the A, B, AB, and 0 subgroups), white blood cells), prostate, cornea, fetal organs, and combinations thereof).

Reappearance of MHC antigens may be used to advantage for inducing tolerance to the donor graft, e.g., through re-education of the recipient's immune system to recognize and tolerate the donor antigens upon reappearance. Serial grafts from the same donor are contemplated for "pre-tolerization" of a recipient by transplanting a first donor tissue, treated according to this invention, which regenerates MHC class I donor antigens that are exposed to the recipient's immune system. A second donor tissue (treated or untreated) may be transplanted into the recipient host, which is now tolerant of the donor tissue.

Although pre-transplantation treatment of the tissues will be the most common practice, it is also contemplated that the method of the present invention can be employed in situ to effect local immune response inhibition. In such embodiments, the method may be utilized to preserve previously transplanted tissue. In such cases, cleavage of the surface antigen produces a soluble, competitive receptor for the cells of the host's immune system , which may serve to effectively blunt immune attack on the transplant. Furthermore, the method described herein may be employed in vivo to reduce or inhibit a transplant rejection, such as previously described for GVHD or first-set and second set rejections. In vivo treatment of a patient experiencing a transplant rejection may include removal of the organ from the recipient host followed by bathing the organ in a solution (e.g., an organ storage solution, such as Belzer UW solution) containing an enzyme capable of cleaving MHC class I antigens (e.g., a cysteine protease, such as papain (e.g., an activated papain enzyme)), according to the methods of the present invention, and then, optionally, washing the organ prior to returning the organ to the host recipient. Alternatively, the organ exhibiting signs of transplant rejection may, in situ in the recipient host, be bathed in or perfused with a solution (e.g., an organ storage solution, such as Belzer UW solution) containing an enzyme capable of cleaving MHC class I antigens (e.g., a cysteine protease, such as papain (e.g., an activated papain enzyme)). Such treatments as bathing or perfusing the organ with the described solution may be performed for a time period of about 1 minute to about 48 hours, e.g., about 1 hour to about 5 hours (e.g., about 3 hours). Machine perfusion with a solution (e.g., an organ storage solution, such as Belzer UW solution) may also be performed on the recipient host to perfuse the tissue and ensure a uniform distribution of the solution throughout the organ.

Any enzyme that is capable of cleaving MHC class I proteins is suitable for use in the method of this invention, e.g., a proteolytic enzyme (.e.g., a cysteine protease), glycosidase, proteinase, or combination thereof, which may sufficiently alter the surface antigens to inhibit subsequent transplant rejection. Examples of such enzymes include papain (e.g., activated papain, as discussed below) or a papain-like enzyme (e.g., a cathepsin), endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

Preferably, the enzymatic reagent for use in the method of this invention is a cysteine protease. More specifically, the enzyme is papain (e.g., activated papain, as discussed below). Papain is the major ingredient of meat tenderizers and is a sulfhydryl protease isolated from the latex of the green fruit of papaya with a targeted capacity to selectively remove MHC class I proteins from donor human cells. Papain was first isolated in 1955, and the enzymatic capabilities of this protease have been extensively documented. Previous studies have elegantly demonstrated that MHC class I, regardless of species or allele type, is selectively cleaved by papain at a highly conserved exterior site of the protein adjacent to the cell membrane (Figure 1 ). Papain is most active at 37^SC or higher temperatures and dissolved into distilled water. The enzyme is inactive in the native state and may be activated by incubation at a temperature, e.g., of about 37^SC, or in the presence of certain chemicals, e.g., cysteine (about 0.0001 M to about 0.01 M, such as about 0.005M) and/or EDTA (about 0.0001 M to about 0.1 M, such as about 0.002M), and/or a specified pH (e.g., a pH in the range of 7.0-8.5, such as 8.0). The activated enzyme may then be stored by cooling or freezing (e.g., storage at about 0 ^SC to about -80 ^SC, such as about - 20^SC), preferably in the absence of oxygen, such as in an anoxic storage container. Therefore, donor tissue treatments may be advantageously performed with a high degree of control by utilizing native papain in the presence of an activator(s) or papain that has been chemically activated. See, generally, Arnon, R., Methods in Enzymology, 19:226-244 (1970) ; Stockell et al., J. Biol. Chem. 227:1 -26 (1957). Papain may cleave MHC class I proteins on cells, tissues, or whole organs. Papain may be used in combination with other enzymes, such as those described above.

The treatment methods of the present invention are advantageously performed by contacting the donor tissue with a solution containing active enzyme(s), e.g., under conditions and for a period of time sufficient to reduce the level of cell surface MHC class I antigens (e.g., by at least about 10% or more as compared to untreated donor tissue). The exact concentration of enzymes (e.g., about 0.1 μg/ml to about 10 mg/ml, such as 40 μg/ml), the duration of the contacting step, and other conditions, such as temperature, can be adjusted by the practitioner to optimize the desired results. Any reduction of MHC class I antigen on the surface of the donor tissue that results in an attenuated immune response by the host is desirable. In general, a more significant reduction in the surface population of MHC class I antigens will result in a more attenuated host immune response. Where more than one enzyme is used, consideration of the optimal cleavage conditions and rate of the desired enzymatic reaction for each individual enzyme can be used to determine whether the enzymes would be most effectively employed serially or together.

Conditions for preparing donor tissue for transplantation

The donor tissue can be prepared for transplantation by immersion in an organ storage solution.

A goal of organ preservation is to maintain function of the organ and tissue during storage, which may be achieved by the use of solutions that seek to mimic the intracellular electrolyte balance of mammalian cells and reduce the redox changes associated with organ hypoxia during preservation. Organ storage solutions for use in the methods of the invention may be selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1

Solution, and combinations thereof. Belzer-UW Solution is a particularly preferred organ storage solution. An organ storage solution that can be used to assist in preserving cellular tissue function may include, but is not limited to, one or more (e.g., two, three, four, or five or more) of the following components:

potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch, glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, HES, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and PEG. In particular, the organ storage solution contains at least raffinose, lactobionate, adenosine, allopurinol, and glutathione.

Specific organ storage solutions are those that include the following components:

i) potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and hydroxyethyl starch;

ii) glucose, mannitol, sucrose, and potassium ; citrate, HEPES, mannitol, potassium, sodium, and magnesium ;

iii) histidine, mannitol, tryptophan, and alpha-ketoglutarate; sodium, latobionate, mannitol, histidine, and reduced glutathione;

iv) trehalose, gluconate, H ES, N-acetylcysteine, dibutyryl cAMP, and nitroglycerin; and v) PEG, potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and hydroxyethyl starch.

For example, the organ storage solution may contain potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and hydroxyethyl starch (e.g., Belzer-UW solution). Belzer-UW Solution is considered the gold standard for the preservation of liver and other donor tissues and has various advantages over other available solutions, such as the inclusion of metabolically inert substrates, e.g., lactobionate and raffinose. The composition of Belzer- UW Solution renders it an optimal preservation solution for multiple organs. The advantages of Belzer- UW Solution in use include the prevention of edema (raffinose, lactobionate), the maintenance of metabolic activity (via supplementation with a precursor of ATP (adenosine)), and antioxidant defense (allopurinol, reduced glutathione). The donor tissue can be prepared for transplantation by immersion in a bath or perfusion with a solution (e.g., an organ storage solution, such as Belzer-UW Solution) containing an enzyme capable of cleaving MHC class I antigens (e.g., a cysteine protease, such as papain (e.g., an activated papain enzyme)). Perfusion may also be performed on the entire tissue or organ donor (e.g., mammal, such as human). Machine perfusion with the organ storage solution (e.g., Belzer UW solution) may also be performed on the donor tissue to ensure a uniform distribution of the solution throughout the tissue.

The present invention is especially directed to the reduction of donor MHC class I antigens through the combination of enzymatic treatment, e.g. with a cysteine protease (e.g., papain) or other enzyme described herein, in which the enzyme is present in an organ storage solution, e.g., Belzer-UW Solution or other organ storage solution described herein.

We have discovered, in particular, that papain retains proteolytic activity and effective cleaving of the MHC class I antigens in an organ storage solution, such as Belzer-UW solution, instead of distilled water. Papain is shown to be soluble in organ storage solution, such as Belzer-UW solution, and exhibits proteolytic activity comparable to that observed in distilled water when incubation periods are extended, which was an unexpected result because of the relatively high sodium content of Belzer-UW Solution. Surprisingly, papain also exhibits comparable proteolytic activity across a broad temperature range including low temperature incubations, e.g., 4^SC. Following enzymatic activation, as described above, papain was successful in cleaving MHC class I antigens at low temperature conditions that are typical of organ preservation, with longer incubation periods that are suitable for preservation.

In an example, the donor tissue (e.g., a whole or partial organ) can be contacted with an organ storage solution, such as Belzer-UW Solution, that includes papain. Treatment of the donor tissue can be for about 1 minute to about 48 hours (e.g., about 1 hour to about 5 hours, such as about 3 hours). The treatment may occur at a temperature in the range of about 4Ό to about 40 °C, such as a temperature of about 37Ό or less, about 22°C or less, or about 4°C. For example, the treatment may occur at room temperature. In an example, the donor tissue (e.g., an organ) is contacted with Belzer-UW Solution that includes papain for about 1 hour to about 5 hours (e.g., about 3 hours) at a temperature in the range of about 4°C to about 40 °C, such as a temperature of about 37Ό or less or 22 °C or less.

After treatment with the enzyme, the donor tissue may be transplanted into a host recipient, e.g., within a period of about 1 minute to about 48 hours after treatment with the enzyme, e.g., about 1 hour to about 5 hours (e.g., about 3 hours) after treatment with the enzyme.

After enzyme treatment, and prior to tissue transplantation, the donor tissue may also be perfused with a solution. Examples of perfusing liquids include or contain one or more of the following: saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, therapeutic agents, and combinations thereof. If desired, the enzyme (e.g., papain) may be added to the perfusing liquid. The perfusing liquid may be removed from the donor tissue by washing prior to tissue transplantation. Alternatively, the transplant tissue may be transplanted directly from the perfusing liquid. In some embodiments, donor tissue may be transplanted within a period of about 1 minute to about 48 hours, e.g., about 1 hour to about 5 hours (e.g., about 3 hours) , after bathing or perfusing in the perfusing liquid.

Also, transplantation packs are contemplated for shipment or storage of donor cells, tissues or organs. The transplantation packs can include a container enclosing together the donor tissue with a preservative or nutrient solution for maintaining the viability of the donor tissue. One or more enzymes (e.g., papain) capable of cleaving MHC class I antigens on the donor tissue may be included in the preservative or nutrient solution. Alternatively, the donor tissue intended for transplant can be briefly treated immediately prior to transplantation into the host, e.g., in the operating room . The donor tissue can also be connected to a machine capable of continuously circulating a preservative or nutrient solution prior to transplantation. If desired, an enzyme (e.g., papain) capable of cleaving MHC class I antigens on the donor tissue may be included in the preservative or nutrient solution during this procedure.

Once a donor tissue has been prepared according to the methods described above, the donor tissue may be administered to, or transplanted into, a host recipient according to methods known in the art.

Other target structures

Although removal of MHC class I antigens has been specifically described, there are many cell- cell interactions that may be undesirable, not only in the context of transplant rejection but also in infection, conception, inflammation, allergy, and many other physiological phenomena. For example, the activation of CTLs touching off the lytic cascade that leads to the transplantation reaction, not only involves binding of the CTL T cell receptor with the MHC class I antigen on the target cell but also involves reciprocal interaction of CD8 with the MHC class I proteins, CD2 with LFA-3, and LPA-1 with ICAM-1 . See, e.g., Faustman et al., Science, 252:1700-2 (1991 ). Cleavage of surface LFA-3 and/or ICAM-1 , or surface adhesion molecules involved with other pathways than the cytolytic pathway, may also be accomplished following the principles of this disclosure, thereby heading off additional avenues of immune attack, or other undesirable cell-cell interactions. After analysis of the known target structures, many well-characterized enzymes can be used to remove or alter these structures so as to further attenuate the reactivity of the structures with its natural receptor(s).

Additional enzymatic reagents that can be used to prepare donor tissue for transplantation include oxidoreductases acting on: (1 ) OH— OH groups; (2) aldehyde or keto groups; (3) CH— CH groups; (4) CH— NH2 groups; (5) reduced AN D or NADP; (6) nitrogenous compounds; (7) diphenols; (8) acting on H20; (9) hydrogen; (10) acting on single donors with incorporation of oxygen; and (1 1 ) acting on paired donors with incorporation of oxygen into one donor; transferases: (1 ) transferring one-carbon groups (methyltransferase, hydroxymethyl-, formyl- and related transferases, carboxyl- and

carbarnoyltransferases, amidinotransferases) ; (2) transferring aldehydic or ketonic residues; (3) acting on acyltransferases (acyltransferases, aminoacyltransferases) ; (4) acting on glycosyltransferases

(hexosyltransferases, pentosyltransferases) ; (5) transferring alkyl or related groups; (6) transferring nitrogenous groups; (7) transferring phosphorus-containing groups (phosphotransferases with an alcohol group as acceptor, phosphotransferases with a carboxyl group as acceptor, phosphotransferases with a nitrogenous group as acceptor, phosphotransferases with a phosphate group as acceptor,

phosphortransferases, pyrophosphotransferases,, nucleotidyltransferases, transferases for other substituted phospho-groups) ; and, (8) transferring sulfur-containing groups (sulfurtransferases, sulfotransferases, CoA-transferases) ; hydrolases: (1 ) acting on ester bonds (carboxylic ester hydrolases, thiolester hydrolases, phosphoric monoester hydrolases, phosphoric diester hydrolases, triphosphoric monoester hydrolases, sulfuric esterhydrolases) ; (2) acting on glycosyl compounds (glycoside hydrlases, hydrolysing N-glycosyl compounds, Hydrolysing S-glycosyl compounds) ; (3) acting on ether bonds (thioether hydrolases) ; (4) acting on peptide bonds (peptide hydrolases) (a-amino-acyl-peptide hydrolases, peptidyl-amino-acid hydrolases, dipeptide hydrolases, peptidyl-peptide hydrolases) ; (5) acting on C— N bonds other than peptide bonds (in linear amides, in cyclic amides, in linear amidines, in cyclic amidines, in cyanides) ; (6) acting on acid-anhydride bonds (in phosphoryl-containing anhydrides) ; (7) acting on C=C bonds; (8) acting on carbon-halogen bonds; and (9) acting on P— N bonds; lyases (1 ) acting on carbon-carbon bonds (carboxyl-lyases, aldehyde-lyases, keto acid-lyases) ; (2) acting on carbon-oxygen bonds (hydrolases and other carbon-oxygen lyases) ; (3) acting on carbon-nitrogen bonds (amonia-lyases and amidine-lyases) ; (4) carbon-sulfur lyases; (5) carbon-halogen lyases; and (6) other lyases; isomerases: (1 ) racemases and epimerases (acting on amino acids and derivatives; acting on hydoxyacids and derivatives, acting on carbohydrates and derivatives, acting on other compounds) ; (2) acting on cis-trans isomerases; (3) acting on intramolecular oxidoreductases (interoconverting aldoses and ketoses, interconverting keto- and enol-groups, transposing C=C bonds) ; (4) acting on intramolecular transferases (transferring acyl groups, transferring phosphoryl groups, transferring other groups) ; (5) acting on intramolecular lyases; and (6) other isomerases; ligases: (1 ) acting on forming C— 0 bonds (amino-acid-RNA ligases) ; (2) acting on forming C— N bonds (acid-ammonia ligases (amide synthetases), acid-amino-acid ligases (peptide synthetases), cyclo-ligases, other C— N ligases, C— N ligases with glutamine as N-donor) ; and (3) forming C— C bonds; and glycosidases, such as a-mylase, β-amylase, glucoamylase, celulase, laminarinase, inulase, dextranase, chitinase, polygalacturonase, lysozyme, neuraminidase, a-glucosidase, β-glucosidase, a-galactosidase, β-galactosidase, a-mannosidase, β- fructofuranosidase, trehalase, chitobiase, β-acetylglucosaminidase, β-glucuronidase, dextrin-1 ,6- glucosidase, hyaluronidase, β-D-fucosidase, metalopeptidases and nucleosidase.

Kits of the invention

The present invention also features kits for preparing donor tissue for transplantation that contain an enzyme that cleaves MHC class I antigens and an organ storage solution. In general, the kits may include one or more containers filled with one or more of the reagents of the invention (e.g., the enzyme and the organ storage solution). The enzyme may be present in the kits in an inactive form, or it may be present in an activated form (e.g., activated papain prepared as described above). Alternatively, the kits may include the necessary reagents (e.g., cysteine and/or EDTA) for incubating the enzyme (e.g., papain) to produce the active form . Such kits can be used in a diversity of transplant settings and have the potential to reduce the need to administer immunosuppressive drugs to the transplant patient or to reduce the dose of immunosuppressive drugs administered to the transplant patient.

Examples

The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed modified to yield essentially the same or similar results.

Example 1 : Materials and methods

Studies of the proteolytic activity of papain in Belzer-UW Solution

To understand the kinetics of papain activity, an in vitro enzymatic assay on a test substrate, casein, was first studied for adapting papain to transplant conditions. This allowed the enzymatic activity of papain to be studied in distilled water (DW) compared to Belzer-UW solution, 4^SC versus 37^SC incubation temperature, prior to the identification of optimal conditions for testing on viable cells.

Casein is a test protein that can be cleaved with activated papain. The cleavage of casein in solution changes the optical reflections of the solution that can be read as a change in absorbance of the solution at 280 nm , the optical density (OD). The results are corrected for blank solutions with no papain. The results are plotted against papain concentrations. This calibration curve is not linear but based on temperature and length of activation time allowed for papain activity.

The regular or standard method for the activation of papain for in vitro assays of chemical activity is to place crystalline papain (Sigma, St. Louis, MO) in distilled water (DW) (Invitrogen, Grand Island, NY) at 23^SC (room temperature, RT) with cysteine and EDTA (conditions described below). Casein, the test substrate, is typically placed in distilled water and then heated for 15 minutes in boiling water to bring about complete dissolution. The standard test assay of papain activity next involves the dissolved substrate casein being added to the activated papain with the reaction continued at a temperature of at least 37^SC. Although these might be ideal conditions for papain activation, heating organs to 37^SC or exposing organs or cells to distilled water is not compatible with organ survival. Here we compared papain activity in DW versus Belzer-UW Solution (Bridge To Life, Columbia, SC) and also refine papain cutting activity by attempting to identify the length of time papain must be added to the substrate if the temperature is lowered to favored transplantation conditions of 4°C.

To display enzymatic activity, papain first requires activation. The activating agents include freshly prepared solution of 0.05M Cysteine (Sigma, St. Louis, MO) + 0.02M Ethylenediaminettraacetic acid (EDTA) (Sigma, St. Louis, MO), adjusted to a pH=8.0 with the desired papain crystals added to the solution to a final high concentration of 0.05mg/ml. Papain has excellent solubility so it is possible to dissolve papain as even a high stock concentrates if needed. Under the standard method to study papain kinetics, which will be referred to here as the Regular Protocol, the activated papain solution is then added to the substrate casein at 37^SC to start the assay (Figure 1 ).The mixed solutions were next incubated in distilled water at 37^SC for 10 min. This method results in rapid action by papain, but lacks applicability to whole organ transplantation conditions due to the distilled water and the temperature of 37°C.

Here we test papain activity using a series of modified enzymatic protocols that were adapted to more closely mimic the transplantation operating room and organ handling conditions (Figure 1 B). For instance the substrate casein solution was kept at 4^SC prior to the assay. The papain was dissolved in Belzer-UW solution instead of distilled water. Also to ensure the papain could be dissolved and activated prior to co-incubation with any substrate i.e. casein or cells expressing HLA class I, the papain activating agents were incubated at 37^SC for 10 min, and then were cooled at 4^SC for 10 min to take the heat out of solution. Then, all conditions of papain activity were studied at 4^SC. For example, the activated papain solution was added to the cooled casein solution. Mixed solutions were incubated at 4^SC for 1 hr or 3hrs, at which point the reaction was terminated for an evaluation of efficiency. The concentration of the split products in the supernatant solution was determined by measuring the absorbency of the solution at 280nm. Peripheral blood lymphocyte (PBL) isolation and staining for HLA class I

Human blood specimens served as normal blood lymphocytes for this study through a human studies protocol approved by the Massachusetts General Hospital Human Studies Committee (MGH- 2001 P001379). This approval involves informed consent for all subjects. All blood was drawn and processed within 2 hours to ensure that the isolated human lymphocytes were fresh and not altered by transport or storage conditions. 18 mL of EDTA-treated peripheral blood was laid over 18 mL of Ficoll- PaquePLUS 1 .078g/m (GE Healthcare Bio-Sciences, Uppsala, Sweden). Tubes were then centrifuged at 500 x g for 20 minutes. An individual band was removed from the gradient. The fraction was washed twice with phosphate buffered saline (PBS) (Gibco, Grand Island, NY), pH = 7.2. The concentration of the leukocyte suspension was determined using a Thomas counting chamber. Viability was checked using the trypan blue exclusion test.

Enzymatic removal of MHC class I of human PBLs

To test activation of papain activity for removal of HLA class I from PBLs, the papain was not only placed in Belzer-UW, but activated at 37^SC with EDTA and cysteine, and then the activated papain was cooled to 4^SC prior to contact with fresh human peripheral blood lymphocytes (PBLs). Under the Regular Protocol, isolated PBLs were incubated at 37^SC during the assay. The activated papain solution in Belzer-UW was added to PBLs and the specimens incubated at 37°C for 10 min. Under the modified protocol, isolated PBLs were incubated at 4^SC with the papain at the same temperature. The papain solution and the activating agents were incubated at 37^SC for 10min, and then were cooled at 4°C for

10min to remove the heat from the solution. The papain solution and the activating agents were added to the cooled PBLs and the specimens were incubated in Belzer-UW solution at 4^SC for 1 hr or 3hrs. The evaluation of enzymatic removal of HLA class I structures was performed using a flow cytometer (FACS- Calibur, Becton-Dickinson, SanJose, CA). Monoclonal antibody of HLA class I (W6/32)-FITC (Invitrogen, Carlsbad, CA) was used.

Studies of the one-way mixed lymphocyte response of PBLs to untreated and papain treated allogeneic PBLs

A one-way mixed lymphocyte reaction (MLR) was performed between PBLs from unrelated donors mixed at a one to one ratio with papain treated and untreated stimulator cells. The stimulator cells were inactivated with mitomycin c before being added to the MLR so the responder cells could be studied for their response to cells with diminished HLA class I. The stimulator cells pretreated with papain had 50-60% diminished HLA class I expression. The reaction was performed by seeding 1 x10⁷ PBL into falcon flasks with 1 x10⁷ allogeneic stimulator cells with the final volume of 25ml. Lymphoproliferation was measured at day 5 of co-culture with the use of 5 uCi of [3H]thymidine to each flask for 6 hours.

Statistical analysis

All data analyses were performed by the paired Student t test using GraphPad Prism-5 software (GraphPad Software, Inc., La Jolla, CA). Example 2: Papain has enzymatic activity in Belzer-UW solution

We first show here that papain is capable of cleaving the test substrate, a soluble class I analogue, when dissolved in Belzer-UW solution. To test papain activity in Belzer-UW solution, we set up cutting activity assays used by the enzymology literature to quantify papain activity against the test substrate casein. Casein, like HLA class I, is cleaved by activated papain and the fragments are measured in solution. The measuring of optical density (OD) by the cleavage of casein with papain allows quantification of enzyme efficiency in distilled water compared to Belzer-UW solution and at different incubation temperatures that could reflect the organ preservation needs of the transplantation community.

Figure 2 shows a dose-response curve of papain, with increasing concentrations of casein cleavage products associated with increasing papain concentrations. Papain dissolved in Belzer-UW solution was nearly as active across a range of papain concentrations as was papain dissolved in distilled water. Therefore, papain retains cleavage activity in Belzer-UW solution. For this experiment, the incubations were performed at 37^SC (Figure 1 B, Figure 2).

Example 3: Papain activity at 4²C is slower than 37²C, but corrected by longer incubation times

Papain also retains its enzymatic activity when cooled to 4^SC as long as the incubation time is extended to 3 hrs. This longer reaction time is still within the typical range of time that whole organs undergo storage and transport. Crystalline papain needs to be activated in order to exhibit enzymatic activity at 23^SC or 37^SC, typically dissolved in distilled water. The activation of papain involves adding a small amount of a reducing agent like cysteine combined with the heavy metal binding agent EDTA, all performed at 23^SC or 37^SC. The activated papain is then typically used in the regular protocol at 37^SC with the substrate casein and with the reaction taking 10 minutes (Figure 3).

To study the impact of temperature on papain activity, we activated papain at 37^SC for the prescribed 10 minutes, but then cooled the activated papain to 4^SC to mimic transplant conditions. It is known that papain can be stable in solution for long periods of time if not exposed to air, but we added the variables of lowered temperature and storage. We also wanted to see if this cooled papain would work on casein at lower temperatures. These experiments tested papain cleavage activity on casein in distilled water (Figure 3A) or with Belzer-UW (Figure 3B) but then cooling the activated papain to 4^SC for exposures with casein for 1 hour or 3 hours (Figure 1 B).

We first performed the experiments in distilled water. Figure 3A shows that activated papain cooled to 4^SC can efficiently cleave casein in a dose-response manner. Either a 1 hr casein incubation time or a 3 hr incubation time was similar to the Regular Protocol that was run at 37^SC for only 10 minutes. Since transplant organs typically are perfused for hours during transport, the extra time needed to achieve equivalence of cleavage by papain is still applicable to the transplant setting. The data in

Figure 3B, which was obtained in Belzer-UW solution, reinforced the findings. We found that the Regular Protocol (distilled water, 23^SC activation, and incubation) can be nearly mimicked by papain incubation in Belzer-UW solution for 3 hr at 4^SC. Figure 3B also shows that papain in Belzer-UW solution exhibits activity against casein even if the incubation time is only 1 hr. Example 4: On human cells, papain cleaves off HLA class I in a dose-dependent manner in Belzer- UW solution

Papain also works well to remove HLA class I structures in a dose-response manner with no loss of cell viability after 3 hours of Belzer-UW co-incubation. To ensure that the H LA class I removal by papain yielded a cellular population of PBLs with a lessoned chance for an allograph rejection response, the mixed lymphoyte reaction (MLR) response was studied (Table 1 ). It is also possible that papain's removal of HLA class I structures could enable use of less closely matched organs, which is important considering organ shortages. The in vitro MLR data show that the foreignness of the tissue, at least as it relates to allogeneic HLA class I, is improved by papain pre-treatment.

Table 1 .

MLR proliferative response of PBLs to allogeneic untreated or papain-treated stimulator cells

[3H]TdR incorporation (c.p.m.) [3H]TdR incorporation (c.p.m .)

Responder vs.

Untreated stimulator

stimulator PBLs Papain-treated stimulator

A vs. B 35,372±1 ,273 5,038±328

C vs. D 61 ,00113,521 6,754±126

E vs. F 52,17812,301 2,354±288

G vs. H 41 ,1 1511 ,6874 2,004±199

All the previous experiments show that papain enzymatic activity can be used under whole organ transplant conditions relying on the Belzer-UW solution and temperature of 4°C when exposed to the substrate. The above experiments also show that papain retains activity if it is activated at its preferred temperature of 23^SC or 37^SC and then cooled to 4^SC prior to contact with the substrate. All the aforementioned steps were also successful when combined with Belzer-UW as the activation solution for the papain. The statistics related to these experiments are presented in Table 2.

Table 2.

We next tested papain activity on freshly prepared human lymphocytes to determine its enzymatic capacity to remove HLA class I structures under transplant conditions (Figure 1 B, Figure 4). For these experiments, all papain activation was done in Belzer-UW at 37^SC for 1 0 minutes and then the papain was stored at 4^SC. As Figure 4A and Figure 4B illustrate, human lymphocytes incubated with activated papain and incubated with papain at 4^SC for 3 hrs have a quantifiable and dose-response removal of HLA class I from the cell surface (Figure 4A, Figure 4B). In other words, papain succeeded at cleaving HLA class I structures as efficiently at 4^SC as at 37^SC, when the incubation time was extended to 3 hours. The statistics related to these experiments are presented in Table 1 . The removal of HLA class I from the cell surface was measured by flow cytometry studies with a HLA class I antibody (Figure 4B). The short, 1 hour incubation of fresh human cells with papain at 4^SC, while effective, was less optimal relative to the longer incubation times (Figure 4A).

The flow diagrams of the dose-response of HLA class I removal from PBLs are shown in Figure 4B. The gradual, smooth and quantifiable removal of class I structures from intact cells is illustrated in these panels. The PBLs after the 3 hour exposure to Belzer-UW solution with papain were also studied for viability and in all cases, the cells maintained viability of >98%. To ensure that the H LA class I removal by papain yielded a cellular population of PBLs with a reduced allograph rejection response, the mixed lymphoyte reaction (MLR) response was studied (see Table 1 ). In the one-way MLR, the data in Table 1 shows the proliferative response of PBLs to allogeneic PBLs from four different donors that served as the stimulator cells. The stimulator cells were either untreated or pre-treated immediately before with papain to remove 50-60% of foreign HLA class I prior to pre-culture as described in the Methods. The data shows, the normal brisk proliferative MLR response was significantly inhibited by papain pre-treatment of the stimulator cells to diminish the expression of foreign H LA class I. The proliferative response that continues to express likely represents the allogeneic HLA class I I proteins that are not removed by the papain treatment.

Other Embodiments

All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference herein in their entirety. Various modifications and variations of the described device and methods of use of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

Claims

1 . A method for preparing donor tissue for transplantation comprising contacting said donor tissue at a temperature of 22^SC or less with a composition comprising an enzyme that cleaves MHC class I antigens from the surface of said donor tissue.

2. The method of claim 1 , wherein said contacting reduces or inhibits activation or stimulation of immune cells, preferably wherein said cells are T cells.

3. The method of claim 1 , wherein said contacting reduces or inhibits transplant rejection of said donor tissue by a host recipient.

4. The method of claim 3, wherein said transplant rejection is selected from the group consisting of hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, and graft versus host disease (GVHD).

5. The method of claim 3 or 4, wherein said method reduces or inhibits transplant rejection in said host recipient when said host recipient has a human leukocyte antigen (HLA) type that does not match the HLA type of the donor tissue, preferably wherein said host recipient shares at least 4/6 HLA alleles with said donor tissue.

6. The method of any one of claims 1 to 5, wherein said enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof.

7. The method of any one of claims 1 to 6, wherein said enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu- specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N- chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

8. The method of claim 7, wherein said enzyme is papain.

9. The method any one of claims 1 to 8, wherein said transplantation is an autograft, syngeneic graft, allograft, or xenograft.

10. The method of any one of claims 1 to 8, wherein said donor tissue is from a mammal that is the same species as said host recipient.

1 1 . The method of any one of claims 1 to 8, wherein said donor tissue is from a mammal that is of a different species than said host recipient.

12. The method of any one of claims 1 to 1 1 , wherein said donor tissue is from a human.

13. The method of any one of claims 1 to 1 1 , wherein said host recipient is a human.

14. The method of any one of claims 1 to 13, wherein said donor tissue is genetically modified prior to transplantation.

15. The method of any one of claims 1 to 14, wherein said donor tissue comprises a cell, a tissue, or an organ.

16. The method of claim 15, wherein said organ is a whole or partial organ.

17. The method of claim 15, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet), hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, bone cell, hepatocyte, hematopoietic cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof.

18. The method of claim 15, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

19. The method of claim 15, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

20. The method of claim 15 or 16, wherein said organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

21 . The method of any one of claims 1 to 20, wherein prior to said contacting said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

22. The method of claim 21 , wherein said enzyme is activated in the presence of a reducing agent.

23. The method of claim 22, wherein said reducing agent is cysteine.

24. The method of any one of claims 21 to 23, wherein said enzyme is activated in the presence of EDTA.

25. The method of any one of claims 1 to 24, wherein said contacting occurs at a temperature in the range of about 0^SC to about 15^SC.

26. The method of claim 25, wherein said temperature is about 4^SC.

27. The method of any one of claims 1 to 26, wherein said donor tissue is contacted with said enzyme for a period of about 5 minutes to about 24 hours.

28. The method of claim 27, wherein said donor tissue is contacted with said enzyme for a period of about 1 hour to about 5 hours.

29. The method of claim 28, wherein said donor tissue is contacted with said enzyme for a period of about 3 hours.

30. The method of any one of claims 1 to 29, wherein said composition comprises an organ storage solution.

31 . The method of claim 30, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

32. The method of claim 30 or 31 , wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

33. The method of claim 32, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and hydroxyethyl starch.

34. The method of claim 33, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

35. The method of claim 34, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

36. The method of any one of claims 30 to 35, wherein said enzyme retains activity in said solution.

37. The method of any one of claims 1 to 36, wherein said method further comprises the step of transplanting said donor tissue into said host recipient, preferably wherein said transplanting occurs before MHC class I antigens are re-expressed on the surface of said donor tissue.

38. The method of claim 37, wherein said transplanting occurs within a period of about 1 minute to about 48 hours after said contacting.

39. The method of claim 38, wherein said transplanting occurs within a period of about 1 hour to about 5 hours after said contacting.

40. The method of any one of claims 1 to 39, wherein said method further comprises bathing or perfusing said donor tissue with a perfusing liquid.

41 . The method of claim 40, wherein said bathing or perfusing occurs prior to transplantation.

42. The method of claim 40 or 41 , wherein said perfusing liquid is or comprises saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, or therapeutic agents, or combinations thereof.

43. The method of any one of claims 1 to 42, wherein said method further comprises the step of washing said donor tissue prior to transplanting said donor tissue into said host recipient.

44. The method of any one of claims 1 to 43, wherein said method further comprises the step of transplanting a second donor tissue into said host recipient.

45. The method of claim 44, wherein said second donor tissue is prepared for transplantation by contacting said second donor tissue with an enzyme that cleaves MHC class I antigens from the surface of said second donor tissue.

46. The method of claim 45, wherein said contacting is at a temperature of about 22^SC or less.

47. The method of claim 37, wherein said method further comprises administering an

immunosuppressive drug or an immunomodulatory cell to said host recipient, wherein preferably said immunomodulatory cell is a Treg.

48. The method of claim 47, wherein said immunosuppressive drug or immunomodulatory cell is administered before, simultaneously with, or after said transplantation.

49. The method of claim 47 or 48, wherein said immunosuppressive drug is selected from the group consisting of cyclosporin A, tacrolimus, sirolimus, OKT3, a corticosteroid, daclizumab, basiliximab, azathioprene, mycophenolate mofetil, methotrexate, 6-mercaptopurine, anti-T cell antibodies, cyclophosphamide, leflunamide, brequinar, ATG, ALG, 15-deoxyspergualin, LF15-0195, and bredinin, and combinations thereof.

50. The method of any one of claims 1 to 49, wherein said contacting is performed in situ in said host recipient.

51 . The method of claim 50, wherein said method inhibits or reduces immune rejection of a previously transplanted donor tissue.

52. A method for preparing donor tissue for transplantation comprising contacting said donor tissue with a composition comprising an organ storage solution and an enzyme that cleaves MHC class I antigens from the surface of said donor tissue.

53. The method of claim 52, wherein said contacting reduces or inhibits activation or stimulation of immune cells, preferably wherein said immune cells are T cells.

54. The method of claim 52, wherein said contacting reduces or inhibits transplant rejection of said donor tissue by a host recipient.

55. The method of claim 54, wherein said transplant rejection is selected from the group consisting of hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, and GVHD.

56. The method of claim 54 or 55, wherein said method reduces or inhibits transplant rejection in said host recipient when said host recipient has a human leukocyte antigen (HLA) type that does not match the HLA type of the donor tissue, preferably wherein said host recipient shares at least 4/6 HLA alleles with said donor tissue.

57. The method of any one of claims 52 to 56, wherein said donor tissue is contacted at a temperature of 40^SC or less.

58. The method of claim 57, wherein said donor tissue is contacted at a temperature in the range of about 22^SC to about 40^SC.

59. The method of claim 58, wherein said donor tissue is contacted at a temperature of about 37^SC.

60. The method of any one of claims 52 to 59, wherein said enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof.

61 . The method of any one of claims 52 to 60, wherein said enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu- specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N- chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

62. The method of claim 61 , wherein said enzyme is papain.

63. The method any one of claims 52 to 62, wherein said transplantation is an autograft, syngeneic graft, allograft, or xenograft.

64. The method of any one of claims 52 to 62, wherein said donor tissue is from a mammal that is the same species as said host recipient.

65. The method of any one of claims 52 to 62, wherein said donor tissue is from a mammal that is of a different species than said host recipient.

66. The method of any one of claims 52 to 65, wherein said donor tissue is from a human.

67. The method of any one of claims 52 to 65, wherein said host recipient is a human.

68. The method of any one of claims 52 to 67, wherein said donor tissue is genetically modified prior to transplantation.

69. The method of any one of claims 52 to 68, wherein said donor tissue comprises a cell, a tissue, or an organ.

70. The method of claim 69, wherein said donor tissue comprises a whole or partial organ.

71 . The method of claim 69, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet), hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, hepatocyte, hematopoietic cell, bone cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof.

72. The method of claim 69, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

73. The method of claim 69, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

74. The method of claim 69 or 70, wherein said organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

75. The method of any one of claims 52 to 74, wherein prior to said contacting said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

76. The method of claim 75, wherein said enzyme is activated in the presence of a reducing agent.

77. The method of claim 76, wherein said reducing agent is cysteine.

78. The method of any one of claims 75 to 77, wherein said enzyme is activated in the presence of EDTA.

79. The method of any one of claims 52 to 78, wherein said donor tissue is contacted with said enzyme for a period of about 5 minutes to about 24 hours.

80. The method of claim 79, wherein said donor tissue is contacted with said enzyme for a period of about 1 hour to about 5 hours.

81 . The method of claim 80, wherein said tissue is contacted with said enzyme for a period of about 3 hours.

82. The method of any one of claims 52 to 81 , wherein said composition comprises an organ storage solution.

83. The method of claim 82, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

84. The method of claim 83, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

85. The method of claim 84, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

86. The method of claim 85, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

87. The method of claim 86, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

88. The method of any one of claims 52 to 87, wherein said enzyme retains activity in said solution.

89. The method of any one of claims 52 to 88, wherein said method further comprises the step of transplanting said donor tissue into said host recipient, preferably wherein said transplanting occurs before MHC class I antigens are re-expressed on the surface of said donor tissue.

90. The method of claim 89, wherein said transplanting occurs within a period of about 1 minute to about 48 hours after said contacting.

91 . The method of claim 90, wherein said transplanting occurs within a period of about 1 hour to about 5 hours after said contacting.

92. The method of any one of claims 52 to 91 , wherein said method further comprises bathing or perfusing said donor tissue with a perfusing liquid.

93. The method of claim 92, wherein bathing or perfusing occurs prior to transplantation.

94. The method of claim 93, wherein said perfusing liquid is or comprises saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, or therapeutic agents, or combinations thereof.

95. The method of any one of claims 52 to 94, wherein said method further comprises the step of washing said donor tissue prior to transplanting said donor tissue into said host recipient.

96. The method of any one of claims 52 to 95, wherein said method further comprises the step of transplanting a second donor tissue into said host recipient.

97. The method of claim 96, wherein said second donor tissue is prepared for transplantation by contacting said second donor tissue with an enzyme that cleaves MHC class I antigens from the surface of said second donor tissue.

98. The method of claim 97, wherein said contacting is at a temperature of about 22^SC or less.

99. The method of any one of claims 52-98, wherein said method further comprises administering an immunosuppressive drug or an immunomodulatory cell to said host recipient, wherein preferably said immunomodulatory cell is a Treg.

100. The method of claim 99, wherein said immunosuppressive drug or immunomodulatory cell is administered before, simultaneously with, or after transplantation of said donor tissue to said host recipient.

101 . The method of claim 99 or 1 00, wherein said immunosuppressive drug is selected from the group consisting of cyclosporin A, tacrolimus, sirolimus, OKT3, a corticosteroid, daclizumab, basiliximab, azathioprene, mycophenolate mofetil, methotrexate, 6-mercaptopurine, anti-T cell antibodies, cyclophosphamide, leflunamide, brequinar, ATG, ALG, 15-deoxyspergualin, LF15-0195, and bredinin, and combinations thereof.

102. The method of any one of claims 52 to 101 , wherein said contacting is performed in situ in said host recipient.

103. The method of claim 102, wherein said method inhibits or reduces immune rejection of a previously transplanted donor tissue.

104. A method for reducing or inhibiting a immune rejection comprising contacting donor tissue at a temperature of about 22^SC or less with an enzyme that cleaves MHC class I antigens from the surface of said donor tissue, wherein preferably said immune rejection is hyperacute rejection, acute vascular rejection, chronic rejection, rejections due to non-adherence, or graft versus host disease (GVHD).

105. A method for treating GVHD in a host recipient in need thereof comprising contacting transplanted donor tissue in situ in said host recipient with an enzyme that cleaves MHC class I antigens from the surface of said donor tissue.

106. The method of claim 105, wherein said host recipient exhibits one or more symptoms of GVHD selected from the group consisting of abdominal pain, cramping, diarrhea, fever, jaundice, skin rash, vomiting, weight loss, dry eyes, dry mouth, hair loss, hepatitis, lung disorders, and digestive tract disorders.

107. A method for reducing or inhibiting immune rejection comprising transplanting a donor tissue treated according to the method of any one of claims 1 to 1 03 into a host recipient.

108. The method of claim 107, wherein said donor tissue comprises a cell, wherein preferably said cell is administered by parenteral administration, oral administration, subcutaneous administration, intravenous administration, intraarterial administration, or topical administration.

109. A composition for preparing donor tissue for transplantation comprising an enzyme that cleaves MHC class I antigens and an organ storage solution.

1 10. A composition for transplantation comprising donor tissue, an enzyme that cleaves MHC class I antigens, and an organ storage solution.

1 1 1 . The composition of claim 109 or 1 10, wherein the enzyme is a proteolytic enzyme, glycosidase, proteinase, or a combination thereof.

1 12. The composition of any one of claims 109-1 1 1 , wherein the enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N- chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

1 13. The composition of claim 1 12, wherein the enzyme is papain.

1 14. The composition of any one of claims 109 to 1 13, wherein said donor tissue is from a mammal.

1 15. The composition of any one of claims 109 to 1 14, wherein said donor tissue is from a human.

1 16. The composition of any one of claims 109 to 1 15, wherein said donor tissue is genetically modified.

1 17. The composition of any one of claims 109 to 1 16, wherein said donor tissue comprises a cell, a tissue, or an organ.

1 18. The composition of claim 1 17, wherein said organ is a whole or partial organ.

1 19. The composition of claim 1 17, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet) , hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, bone cell, hepatocyte, hematopoietic cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), a brain cell (e.g., a neuron, astrocyte, and meningeal

cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof.

120. The composition of claim 1 17, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

121 . The composition of claim 1 17, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

122. The composition of claim 1 17 or 1 18, wherein said organ is selected from the group consisting of nervous system , brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear,

hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

123. The composition of any one of claims 109 to 122, wherein said enzyme is an activated enzyme.

124. The composition of claim 123, wherein said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

125. The composition of claim 124, wherein said enzyme is activated in the presence of a reducing agent.

126. The composition of claim 125, wherein said reducing agent is cysteine.

127. The composition of any one of claims 123 to 126, wherein said enzyme is activated in the presence of EDTA.

128. The composition of any one of claims 109 to 127, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

129. The composition of claim 128, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

130. The composition of claim 129, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

131 . The composition of claim 130, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 1 0 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

132. The composition of any one of claims 109 to 131 , wherein said enzyme retains activity in said solution.

133. A kit for preparing donor tissue for transplantation, said kit comprising an enzyme that cleaves MHC class I antigens from the surface of said donor tissue and an organ storage solution.

134. The kit of claim 133, wherein said enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof.

135. The kit of claim 134, wherein said enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, or endopeptidase, or a combination of such enzymes.

136. The kit of claim 135, wherein said enzyme is papain.

137. The kit of any one of claims 133-136, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

138. The kit of claim 137, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

139. The kit of claim 138, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

140. The kit of claim 139, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

141 . The kit of claim 140, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

142. The kit of any one of claims 133 to 141 , wherein said enzyme retains activity in said solution.

143. The kit of any one of claims 133 to 142, wherein said enzyme and said organ storage solution are in a container in said kit.

144. The kit of claim 143, wherein said enzyme and said organ storage solution are in the same container.

145. The kit of claim 144, wherein said enzyme and said organ storage solution are in different containers.

146. The method of claim 3, wherein said method reduces or inhibits transplant rejection in said host recipient when said host recipient has a human leukocyte antigen (HLA) type that does not match the HLA type of the donor tissue, preferably wherein said host recipient shares at least 4/6 HLA alleles with said donor tissue.

147. The method of claim 1 , wherein said enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof.

148. The method of claim 1 , wherein said enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

149. The method of claim 148, wherein said enzyme is papain.

150. The method of claim 1 , wherein said transplantation is an autograft, syngeneic graft, allograft, or xenograft.

151 . The method of claim 1 , wherein said donor tissue is from a mammal that is the same species as said host recipient.

152. The method of claim 1 , wherein said donor tissue is from a mammal that is of a different species than said host recipient.

153. The method of claim 1 , wherein said donor tissue is from a human.

154. The method of claim 1 , wherein said host recipient is a human.

155. The method of claim 1 , wherein said donor tissue is genetically modified prior to transplantation.

156. The method of claim 1 , wherein said donor tissue comprises a cell, a tissue, or an organ.

157. The method of claim 156, wherein said organ is a whole or partial organ.

158. The method of claim 156, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet) , hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, bone cell, hepatocyte, hematopoietic cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof.

159. The method of claim 156, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

160. The method of claim 156, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

161 . The method of claim 156, wherein said organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

162. The method of claim 1 , wherein prior to said contacting said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

163. The method of claim 162, wherein said enzyme is activated in the presence of a reducing agent.

164. The method of claim 163, wherein said reducing agent is cysteine.

165. The method of claim 162, wherein said enzyme is activated in the presence of EDTA.

166. The method of claim 1 , wherein said contacting occurs at a temperature in the range of about 0^SC to about 15^SC.

167. The method of claim 166, wherein said temperature is about 4^SC.

168. The method of claim 1 , wherein said donor tissue is contacted with said enzyme for a period of about 5 minutes to about 24 hours.

169. The method of claim 168, wherein said donor tissue is contacted with said enzyme for a period of about 1 hour to about 5 hours.

170. The method of claim 169, wherein said donor tissue is contacted with said enzyme for a period of about 3 hours.

171 . The method of claim 1 , wherein said composition comprises an organ storage solution.

172. The method of claim 171 , wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

173. The method of claim 172, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

174. The method of claim 173, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and hydroxyethyl starch.

175. The method of claim 174, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

176. The method of claim 175, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

177. The method of claim 176, wherein said enzyme retains activity in said solution.

178. The method of claim 1 , wherein said method further comprises the step of transplanting said donor tissue into said host recipient, preferably wherein said transplanting occurs before MHC class I antigens are re-expressed on the surface of said donor tissue.

179. The method of claim 178, wherein said transplanting occurs within a period of about 1 minute to about 48 hours after said contacting.

180. The method of claim 179, wherein said transplanting occurs within a period of about 1 hour to about 5 hours after said contacting.

181 . The method of claim 1 , wherein said method further comprises bathing or perfusing said donor tissue with a perfusing liquid.

182. The method of claim 181 , wherein said bathing or perfusing occurs prior to transplantation.

183. The method of claim claim 181 , wherein said perfusing liquid is or comprises saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, or therapeutic agents, or combinations thereof.

184. The method of claim 1 , wherein said method further comprises the step of washing said donor tissue prior to transplanting said donor tissue into said host recipient.

185. The method of claim 1 , wherein said method further comprises the step of transplanting a second donor tissue into said host recipient.

186. The method of claim 185, wherein said second donor tissue is prepared for transplantation by contacting said second donor tissue with an enzyme that cleaves MHC class I antigens from the surface of said second donor tissue.

187. The method of claim 186, wherein said contacting is at a temperature of about 22^SC or less.

188. The method of claim 178, wherein said method further comprises administering an

189. The method of claim 188, wherein said immunosuppressive drug or immunomodulatory cell is administered before, simultaneously with, or after said transplantation.

190. The method of claim 188, wherein said immunosuppressive drug is selected from the group consisting of cyclosporin A, tacrolimus, sirolimus, OKT3, a corticosteroid, daclizumab, basiliximab, azathioprene, mycophenolate mofetil, methotrexate, 6-mercaptopurine, anti-T cell antibodies, cyclophosphamide, leflunamide, brequinar, ATG, ALG, 15-deoxyspergualin, LF15-0195, and bredinin, and combinations thereof.

191 . The method of claim 1 , wherein said contacting is performed in situ in said host recipient.

192. The method of claim 191 , wherein said method inhibits or reduces immune rejection of a previously transplanted donor tissue.

193. The method of claim 52, wherein said donor tissue is contacted at a temperature of 40^SC or less.

194. The method of claim 193, wherein said donor tissue is contacted at a temperature in the range of about 22^SC to about 40^SC.

195. The method of claim 194, wherein said donor tissue is contacted at a temperature of about 37^SC.

196. The method of claim 52, wherein said enzyme is a proteolytic enzyme, glycosidase, proteinase, or combination thereof.

197. The method of claim 52, wherein said enzyme is papain or a papain-like enzyme, endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu-specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N-chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

198. The method of claim 197, wherein said enzyme is papain.

199. The method of claim 52, wherein said transplantation is an autograft, syngeneic graft, allograft, or xenograft.

200. The method of claim 52, wherein said donor tissue is from a mammal that is the same species as said host recipient.

201 . The method of claim 52, wherein said donor tissue is from a mammal that is of a different species than said host recipient.

202. The method of any one of claim 52, wherein said donor tissue is from a human.

203. The method of any one of claim 52, wherein said host recipient is a human.

204. The method of any one of claim 52, wherein said donor tissue is genetically modified prior to transplantation.

205. The method of any one of claim 52, wherein said donor tissue comprises a cell, a tissue, or an organ.

206. The method of claim 205, wherein said donor tissue comprises a whole or partial organ.

207. The method of claim 205, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet) , hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, hepatocyte, hematopoietic cell, bone cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), brain cell (e.g., a neuron, astrocyte, and meningeal cell), chondrocyte, stem cell (e.g., Hox1 1 stem cell), fetal cell, vascular tissue cell, bile duct cell, epithelial cell, endothelial cell, endoderm cell, mesoderm cell, mesenchymal cell, skin cell, and combinations thereof.

208. The method of claim 205, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

209. The method of claim 205, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

210. The method of claim 205, wherein said organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

21 1 . The method of claim 52, wherein prior to said contacting said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

212. The method of claim 21 1 , wherein said enzyme is activated in the presence of a reducing agent.

213. The method of claim 212, wherein said reducing agent is cysteine.

214. The method of claim 21 1 , wherein said enzyme is activated in the presence of EDTA.

215. The method of claim 52, wherein said donor tissue is contacted with said enzyme for a period of about 5 minutes to about 24 hours.

216. The method of claim 215, wherein said donor tissue is contacted with said enzyme for a period of about 1 hour to about 5 hours.

217. The method of claim 216, wherein said tissue is contacted with said enzyme for a period of about 3 hours.

218. The method of claim 52, wherein said composition comprises an organ storage solution.

219. The method of claim 218, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

220. The method of claim 219, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

221 . The method of claim 220, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

222. The method of claim 221 , wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

223. The method of claim 222, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

224. The method of claim 52, wherein said enzyme retains activity in said solution.

225. The method of claim 52, wherein said method further comprises the step of transplanting said donor tissue into said host recipient, preferably wherein said transplanting occurs before MHC class I antigens are re-expressed on the surface of said donor tissue.

226. The method of claim 225, wherein said transplanting occurs within a period of about 1 minute to about 48 hours after said contacting.

227. The method of claim 226, wherein said transplanting occurs within a period of about 1 hour to about 5 hours after said contacting.

228. The method of claim 52, wherein said method further comprises bathing or perfusing said donor tissue with a perfusing liquid.

229. The method of claim 228, wherein bathing or perfusing occurs prior to transplantation.

230. The method of claim 229, wherein said perfusing liquid is or comprises saline, blood, water, antibiotics, analgesics, growth factors, bone marrow aspirates, peptides, cell suspensions, water, antiseptics, clotting agents, proteins, hyrdrogels, hylauronic acid, or therapeutic agents, or combinations thereof.

231 . The method of claim 52, wherein said method further comprises the step of washing said donor tissue prior to transplanting said donor tissue into said host recipient.

232. The method of claim 52, wherein said method further comprises the step of transplanting a second donor tissue into said host recipient.

233. The method of claim 232, wherein said second donor tissue is prepared for transplantation by contacting said second donor tissue with an enzyme that cleaves MHC class I antigens from the surface of said second donor tissue.

234. The method of claim 233, wherein said contacting is at a temperature of about 22^SC or less.

235. The method of claim 52, wherein said method further comprises administering an

236. The method of claim 235, wherein said immunosuppressive drug or immunomodulatory cell is administered before, simultaneously with, or after transplantation of said donor tissue to said host recipient.

237. The method of claim 235, wherein said immunosuppressive drug is selected from the group consisting of cyclosporin A, tacrolimus, sirolimus, OKT3, a corticosteroid, daclizumab, basiliximab, azathioprene, mycophenolate mofetil, methotrexate, 6-mercaptopurine, anti-T cell antibodies, cyclophosphamide, leflunamide, brequinar, ATG, ALG, 15-deoxyspergualin, LF15-0195, and bredinin, and combinations thereof.

238. The method of claim 52, wherein said contacting is performed in situ in said host recipient.

239. The method of claim 238, wherein said method inhibits or reduces immune rejection of a previously transplanted donor tissue.

240. A method for reducing or inhibiting immune rejection comprising transplanting a donor tissue treated according to the method of claim 1 or 52 into a host recipient.

241 . The method of claim 240, wherein said donor tissue comprises a cell, wherein preferably said cell is administered by parenteral administration, oral administration, subcutaneous administration, intravenous administration, intraarterial administration, or topical administration.

242. The composition of claim 109, wherein the enzyme is papain or a papain-like enzyme,

endoproteinase, pepsin, chymotrysin, trypsin, collagenase, cyanogen bromide, enterokinase (Asp or Glu- specific), iodosobenzoate, lysobacter endoproteinase, proleases, N-bromosuccinimide, N- chlorosuccinimide, hydroxylamine, 2-nitro-5-thiocyanobenzoate, endopeptidase, or a combination thereof.

243. The composition of claim 1 10, wherein the enzyme is papain or a papain-like enzyme,

244. The composition of claim 242 or 243, wherein the enzyme is papain.

245. The composition of claim 1 1 1 , wherein the enzyme is papain or a papain-like enzyme,

246. The composition of claim 245, wherein the enzyme is papain.

247. The composition of claim 109 or 1 10, wherein said donor tissue is from a mammal.

248. The composition of claim 109 or 1 10, wherein said donor tissue is from a human.

249. The composition of claim 109 or 1 10, wherein said donor tissue is genetically modified.

250. The composition of claim 109 or 1 10, wherein said donor tissue comprises a cell, a tissue, or an organ.

251 . The composition of claim 250, wherein said organ is a whole or partial organ.

252. The composition of claim 250, wherein said cell is selected from the group consisting of a blood cell (e.g., a red blood cell, white blood cell (e.g., lymphocyte, monocyte, and granulocyte), and platelet) , hepatocyte, cardiac cell, lung cell (e.g., alveolar cell), genetically modified cell, precursor cell, fibroblast, myoblast, bone marrow cell, bone cell, hepatocyte, hematopoietic cell, islet cell (e.g., islets of Langerhans cell), spleen cell (e.g., Hox1 1 + cell), a brain cell (e.g., a neuron, astrocyte, and meningeal

253. The composition of claim 250, wherein said tissue is selected from the group consisting of bone tissue, gastrointestinal tissue, vascular tissue (e.g., blood vessels, such as veins and arteries), bone marrow, islet tissue, cartilage, tendon, skeletal tissue, endothelium, epithelium, smooth muscle, cardiac muscle, skeletal muscle, ocular tissue, glandular tissue, mesenchymal tissue, placental tissue, liver tissue, heart tissue (e.g., heart valve), kidney tissue, colon tissue, bladder tissue, ovarian tissue, testicular tissue, pulmonary tissue, skin tissue, genitourinary tissue, endoderm-derived tissue, mesoderm-derived tissue, ectoderm-derived tissue, and combinations thereof.

254. The composition of claim 250, wherein said tissue is selected from the group consisting of epithelial tissue, nervous tissue (e.g., nerves), connective tissue, muscle tissue, and combinations thereof.

255. The composition of claim 250, wherein said organ is selected from the group consisting of nervous system, brain (e.g., neuronal tissue), spinal cord, heart, lung, kidneys, liver, pancreas, gall bladder, urinary tract, adrenal gland, thymus, spleen, lymph nodes, breast, ovary, testes, uterus, bronchi, eye, nose, pituitary gland, salivary gland, large/small intestine, stomach, thyroid, bone, ear, hypothalamus, larynx, spinal cord, ureter, esophagus, trachea, urethra, hand, face, blood, prostate, cornea, fetal organs, and combinations thereof.

256. The composition of claim 109 or 1 10, wherein said enzyme is an activated enzyme.

257. The composition of claim 256, wherein said enzyme is activated by incubating said enzyme at a temperature in the range of about 22^SC to about 37^SC.

258. The composition of claim 257, wherein said enzyme is activated in the presence of a reducing agent.

259. The composition of claim 258, wherein said reducing agent is cysteine.

260. The composition of claim 256, wherein said enzyme is activated in the presence of EDTA.

261 . The composition of claim 109 or 1 10, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

262. The composition of claim 261 , wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

263. The composition of claim 262, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

264. The composition of claim 263, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 1 0 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

265. The composition of claim 109 or 1 10, wherein said enzyme retains activity in said solution.

266. The kit of claim 133, wherein said organ storage solution is selected from the group consisting of Belzer-UW Solution, Collins Solution, Citrate Solution, Custodiol Solution, Celsior Solution, Kyoto Solution, IGL-1 Solution, and combinations thereof.

267. The kit of claim 266, wherein said organ storage solution comprises one or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, HEPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N- acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG), wherein preferably said organ storage solution comprises two or more components selected from the group consisting of potassium lactobionate, potassium salts, magnesium salts, sodium salts, raffinose, adenosine, glutathione, allopurine, hydroxyethyl starch (HES), glucose, mannitol, sucrose, citrate, H EPES, histidine, tryptophan, alpha-ketoglutarate, trehalose, gluconate, N-acetylcysteine, dibutyryl cAMP, nitroglycerin, and polyethylene glycol (PEG).

268. The kit of claim 267, wherein said organ storage solution comprises potassium lactobionate, potassium phosphate, magnesium phosphate, raffinose, adenosine, glutathione, allopurine, and H ES.

269. The kit of claim 268, wherein said organ storage solution comprises about 10 mM to about 200 mM potassium lactobionate, about .25 mM to about 100 mM potassium phosphate, about .5 mM to about 50 mM magnesium phosphate, about 3 mM to about 100 mM raffinose, about 1 mM to about 50 mM adenosine, about 0.3 mM to 30 mM glutathione, about 0.1 mM to about 10 mM allopurine, and about 1 g/L to about 100 g/L hydroxyethyl starch.

270. The kit of claim 269, wherein said organ storage solution comprises 100 mM potassium lactobionate, 25 mM potassium phosphate, 5 mM magnesium phosphate, 30 mM raffinose, 5 mM adenosine, 3 mM glutathione, 1 mM allopurine, and 50 g/L hydroxyethyl starch.

271 . The kit of claim 133, wherein said enzyme retains activity in said solution.

272. The kit of claim 133, wherein said enzyme and said organ storage solution are in a container in said kit.

273. The kit of claim 272, wherein said enzyme and said organ storage solution are in the same container.

274. The kit of claim 273, wherein said enzyme and said organ storage solution are in different containers.